Receptor tyrosine kinases of the Eph family are upregulated in different types of cancer. EphB4 has been linked to breast cancer and other types of cancer, but little is known about how this receptor may contribute to oncogenesis. The EphB receptors transmit signals via their kinase domain (known as forward signals) and also through the cytoplasmic domain of their transmembrane ephrin-B ligands (known as reverse signals). We found that EphB4 promotes breast cancer cell growth in vivo by stimulating angiogenesis through its ligand, ephrin-B2, which is expressed in tumor blood vessels. This is consistent with the well-established role of EphB4 and ephrin-B2 in blood vessel formation during embryonic development and with the emerging role of these molecules in pathological forms of angiogenesis. Surprisingly, we found that EphB4 forward signaling activity is low in breast cancer cells because of the low levels of ephrin-B2. Furthermore, in vitro stimulation of EphB4 with an ephrin-B2 Fc fusion protein inhibits breast cancer cell survival/proliferation and migration/invasion, suggesting that EphB4 forward signaling inhibits tumorigenesis. These findings are in agreement with a recent report that EphB receptor activity suppresses colorectal cancer progression (Batlle et al., 2005, Nature 435:1126). We have identified a novel Eph signaling pathway responsible for the tumor suppressing effects of EphB4 that involves the cytoplasmic tyrosine kinase Abl and the proto-oncogene Crk. Activation of this pathway by systemic administration of ephrin-B2 Fc inhibits tumor growth in a mouse breast cancer xenograft model. Despite this progress, much remains to be learned about how EphB4 and ephrin-B2 influence tumor progression. Here we propose to use a combination of in vitro cell culture assays and in vivo mouse tumor models to better understand the tumor suppressing and tumor promoting activities of EphB4/ephrin-B2, in order to optimally exploit EphB4 as a breast cancer target. Furthermore, we will develop peptides and small molecules that target EphB4 and evaluate their anti-oncogenic effects in cell culture and in mouse preclinical models. The EphB4-targeting molecules that we propose to develop will also be useful for therapies against vascular diseases involving EphB4 and ephrin-B2. The information obtained will advance our basic understanding of the complex but intriguing molecular mechanisms used by the Eph receptors to influence tumorigenesis and angiogenesis, and possibly lead to new Eph-based therapies.